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Understanding how macrophages promote myocardial repair can help create new therapies for infarct repair. We aimed to determine what mechanisms underlie the reparative properties of macrophages. Cytokine arrays revealed that neonatal cardiac macrophages from the injured neonatal heart secreted high amounts of osteopontin (OPN). In vitro, recombinant OPN stimulated cardiac cell outgrowth, cardiomyocyte (CM) cell-cycle re-entry, and CM migration. In addition, OPN induced nuclear translocation of the cytoplasmatic yes-associated protein 1 (YAP1) and upregulated transcriptional factors and cell-cycle genes. Significantly, by blocking the OPN receptor CD44, we eliminated the effects of OPN on CMs. OPN also activated the proliferation and migration of non-CM cells: endothelial cells and cardiac mesenchymal stromal cells in vitro. Notably, the significant role of OPN in myocardial healing was demonstrated by impaired healing in OPN-deficient neonatal hearts. Finally, in the adult mice, a single injection of OPN into the border of the ischemic zone induced CM cell-cycle re-entry, improved scar formation, local and global cardiac function, and LV remodelling 30 days after MI. In summary, we have shown, for the first time, that recombinant OPN activates cell-cycle re-entry in CMs. In addition, recombinant OPN stimulates multiple cardiac cells and improves scar formation, LV remodelling, and regional and global function after MI. Therefore, we propose OPN as a new cell-free therapy to optimize infarct repair.

Aims One unaddressed aspect of healing after myocardial infarction (MI) is how non-myocyte cells that survived the ischemic injury, keep withstanding additional cellular damage by stress forms typically arising during the post-infarction inflammation. Here we aimed to determine if cell survival is conferred by expression of a mitochondrial protein novel to the cardiac proteome, known as steroidogenic acute regulatory protein, (StAR/STARD1). Further studies aimed to unravel the regulation and role of the non-steroidogenic cardiac StAR after MI. Methods and results Following permanent ligation of the left anterior descending coronary artery in mouse heart, timeline western blot analyses showed that StAR expression corresponds to the inflammatory response to MI. Following the identification of StAR in mitochondria of cardiac fibroblasts in culture, confocal microscopy immunohistochemistry (IHC) identified StAR expression in left ventricular (LV) activated interstitial fibroblasts, adventitial fibroblasts and endothelial cells. Further work with the primary fibroblasts model revealed that interleukin-1α (IL-1α) signaling via NF-κB and p38 MAPK pathways efficiently upregulates the expression of the Star gene products. At the functional level, IL-1α primed fibroblasts were protected against apoptosis when exposed to cisplatin mimicry of in vivo apoptotic stress; yet, the protective impact of IL-1α was lost upon siRNA mediated StAR downregulation. At the physiological level, StAR expression was nullified during post-MI inflammation in a mouse model with global IL-1α deficiency, concomitantly resulting in a 4-fold elevation of apoptotic fibroblasts. Serial echocardiography and IHC studies of mice examined 24 days after MI revealed aggravation of LV dysfunction, LV dilatation, anterior wall thinning and adverse tissue remodeling when compared with loxP control hearts. Conclusions This study calls attention to overlooked aspects of cellular responses evolved under the stress conditions associated with the default inflammatory response to MI. Our observations suggest that LV IL-1α is cardioprotective, and at least one mechanism of this action is mediated by induction of StAR expression in border zone fibroblasts, which renders them apoptosis resistant. This acquired survival feature also has long-term ramifications on the heart recovery by diminishing adverse remodeling and improving the heart function after MI.

Background and Aim: Heart disease might be an independent risk factor for cancer (reverse cardio-oncology). The cellular and molecular mechanisms that link heart disease to cancer remain elusive, and specific therapies are limited. We hypothesized that cardiac extracellular vesicles (cEVs) secreted by diseased hearts carry and disseminate factors that promote tumor growth. Methods & Results: We subjected female mice to myocardial infarction (MI) or sham-MI and 28 days of follow-up. Left ventricular remodeling was confirmed by echocardiography. To determine the role of cEVs in tumor growth, we focused on cardiac mesenchymal stromal cells (cMSCs), which play a central role in cardiac repair, remodeling, and fibrosis. Thus, we isolated cMSCs from mice hearts 10 or 28 days after MI or sham MI. cMSCs after MI secreted more small EVs than cMSCs from sham-MI. Proteomic analysis revealed a distinctive profile of cEVs after MI ( Fig A ). Purified cMSC-EVs targeted both breast and lung cancer cells in vitro. A scratch assay showed that MI-cEVs facilitated cancer cell proliferation and migration two times faster than sham-MI cEVs ( Fig B , p=0.0002). Finally, lung or breast cancer cells were inoculated into the hind limb or mammary pad 10 days before or after MI. Tumor growth was monitored by serial ultrasound examinations. While MI stimulated tumor growth, EV inhibition by GW4869 markedly attenuated this effect ( Fig C ). Conclusions: We show, for the first time, that cMSCs from the infarcted and remodeling heart secret EVs that target tumor cells and facilitate tumor growth. We propose cEVs as potential mediators and therapeutic targets in patients with concomitant heart disease and cancer.

Rapid and sensitive screening tools for SARS-CoV-2 infection are essential to limit the spread of COVID-19 and to properly allocate national resources. Here, we developed a new point-of-care, non-contact thermal imaging tool to detect COVID-19, based on image-processing algorithms and machine learning analysis. We captured thermal images of the back of individuals with and without COVID-19 using a portable thermal camera that connects directly to smartphones. Our novel image processing algorithms automatically extracted multiple texture and shape features of the thermal images and achieved an area under the curve (AUC) of 0.85 in detecting COVID-19 with up to 92% sensitivity. Thermal imaging scores were inversely correlated with clinical variables associated with COVID-19 disease progression. We show, for the first time, that a hand-held thermal imaging device can be used to detect COVID-19. Non-invasive thermal imaging could be used to screen for COVID-19 in out-of-hospital settings, especially in low-income regions with limited imaging resources.HIGHLIGHTSAutomated processing of thermal images of the back can be used to detect COVID-19 with up to 92% sensitivity.The extracted texture features of the thermal image are associated with COVID-19 disease progression and lung injury.A portable thermal camera that connects directly to smartphones can be used to detect COVID-19.Non-invasive thermal imaging could be used to screen for COVID-19 in out-of-hospital settings and regions with limited imaging resources.GRAPHICAL ABSTRACT

Morbidity and mortality from ischaemic heart disease (IHD) and heart failure (HF) remain significant in Europe and are increasing worldwide. Patients with IHD or HF might benefit from novel therapeutic strategies, such as cell-based therapies. We recently discussed the therapeutic potential of cell-based therapies and provided recommendations on how to improve the therapeutic translation of these novel strategies for effective cardiac regeneration and repair. Despite major advances in optimizing these strategies with respect to cell source and delivery method, the clinical outcome of cell-based therapy remains unsatisfactory. Major obstacles are the low engraftment and survival rate of transplanted cells in the harmful microenvironment of the host tissue, and the paucity or even lack of endogenous cells with repair capacity. Therefore, new ways of delivering cells and their derivatives are required in order to empower cell-based cardiac repair and regeneration in patients with IHD or HF. Strategies using tissue engineering (TE) combine cells with matrix materials to enhance cell retention or cell delivery in the transplanted area, and have recently received much attention for this purpose. Here, we summarize knowledge on novel approaches emerging from the TE scenario. In particular, we will discuss how combinations of cell/bio-materials (e.g. hydrogels, cell sheets, prefabricated matrices, microspheres, and injectable matrices) combinations might enhance cell retention or cell delivery in the transplantation areas, thereby increase the success rate of cell therapies for IHD and HF. We will not focus on the use of classical engineering approaches, employing fully synthetic materials, because of their unsatisfactory material properties which render them not clinically applicable. The overall aim of this Position Paper from the ESC Working Group Cellular Biology of the Heart is to provide recommendations on how to proceed in research with these novel TE strategies combined with cell-based therapies to boost cardiac repair in the clinical settings of IHD and HF. © Published on behalf of the European Society of Cardiology. All rights reserved.

Background and aim Epicardial fat (eFat) has been linked to atrial remodeling and fibrillation (AF). Small extracellular vesicles (sEVs) are heterogeneous membrane vesicles released by all cell types. They can both protect and damage tissues by the delivery of multiple different messengers. Surprisingly, the role of sEVs in the pathogenesis of AF has not been studied. Thus, we aimed to determine whether sEVs derived from eFat play a role in the pathogenesis of AF. Methods and results We collected eFat specimens from patients with and without chronic or paroxysmal AF undergoing open-heart surgery. Isolated eFat specimens were cut into small pieces and incubated as organ cultures. We isolated sEVs from the medium of the explant by differential ultra-centrifugation, high-density gradient or size exclusion chromatography (SEC), and characterized vesicle size distribution, morphology, specific markers, histology and molecular cargo. Immunostaining for macrophage accumulation, fibrosis and apoptosis confirmed the pro-inflammatory and pro-fibrotic properties of eFat sEVs from patients with AF (Fig. 1). eFat sEVs labeled with PKH26 were massively up taken by endothelial cells (Fig. 2). Real-time PCR showed an increased level of oxidative stress genes in endothelial cells. eFat sEVs from patients with AF caused more fibrosis after injection into rat hearts than those without AF. (Fig. 3). Finally, while eFat sEVs from patients with and without AF induced shorter action potential duration, only eFat sEVs from patients with AF induced sustained re-entry (rotor) in human induced pluripotent stem cell (iPS)-derived cardiomyocytes (Fig. 4) Conclusion We show, for the first time, that sEVs from eFat of patients with AF demonstrate unique pro-inflammatory, pro-fibrotic and pro-arrhythmic properties. Our findings suggest that eFat sEVs can induce cellular, molecular and electrophysiological remodeling that can subsequently lead to the development of AF. Funding Acknowledgement Type of funding source: None

Background Mesenchymal stromal cells (MSCs) have been implicated in the development and progression of myocardial diseases. However, the role of extracellular vesicles (EVs) secreted from human MSCs (hMSCs) of epicardial fat (eFat) in the pathogenesis of ischemic heart disease (IHD) has never been studied. Purpose We sought to characterize EVs from eFat-hMSCs of patients with and without IHD. Methods We collected eFat specimens from 12 patients with (n=7) and without (n=5) IHD who underwent open heart surgery. We isolated and cultured hMSCs by enzymatic digestion and adherence to plastic. EVs were separated from the culture media by differential ultra-centrifugation. We assessed EV size and structure by electron microscopy and nanoparticle tracking analysis. Finally, we evaluated the proteomic profile of EVs from ischemic and non-ischemic patients by mass spectrometry. Results eFat-hMSCs from ischemic patients, secreted higher amounts of small EVs (size range of 70–200 nm) compared with non-ischemic controls (Figure 1A+B). Proteomic analysis revealed over 1,000 proteins inside isolated EVs. We identified 13 proteins that were significantly differentially expressed between patients with and without IHD, including apolipoprotein E, transforming growth factor-β and collagen α-1 (Figure 1C). We performed an enrichment analysis which showed that EVs from eFat-hMSCs from ischemic patients encapsulated functional protein networks associated with immune activation. Unlike IHD patients, EVs from non-ischemic patients carried functional protein networks that regulate angiogenesis. Conclusions eFat-hMSCs of patients with IHD secrete higher amounts of small EVs with a unique proteomic signature related to immune activation. EVs derived from eFat-hMSCs should be further studied for their potential role in the initiation and progression of cardiovascular diseases, such as coronary artery disease, heart failure and arrhythmias. Figure 1 Funding Acknowledgement Type of funding source: None

Background The environment of the failing and infarcted myocardium drives resident and transplanted mesenchymal stromal cells (MSCs) toward a pro-inflammatory phenotype, thus restricting their survival and their reparative effects in a mechanism mediated by toll-like receptor 4 (TLR4). Hence, new approaches are needed to improve the efficacy of cell therapy for heart failure. CRISPR is a promising tool for genome editing, which raises the hope for therapeutic genome editing in the clinic. Purpose We aimed to provide a new strategy to enhance MSC-based cell therapy to improve cardiac remodeling and function. We hypothesized that ex vivo knockout (KO) of the human TLR4 gene by CRISPR would switch human-cardiac MSCs (hMSCs) to an anti-inflammatory, reparative phenotype that could prevent remodeling of the left ventricle after myocardial infarction (Fig. 1A). Methods and results We achieved up to 68% (out of 4x105 cells, R2=0.93) success rate in editing the genome of primary cardiac hMSCs taken from patients with ischemic heart disease. The deletion of TLR4 in hMSCs significantly reduced the secretion of inflammatory and extracellular-matrix (ECM) proteins, compared with unedited hMSCs, by protein mass spectrometry (Fig. 1B) and by multiplex ELISA (Fig. 1C). Additionally, edited cells secreted significantly more extracellular vesicles (EVs) than unedited hMScs (Fig. 1D, p Conclusions We show, for the first time, that CRISPR-based deletion of the TLR4 gene in hMSCs inhibits inflammatory and ECM protein secretion and facilitates a reparative response by hMSCs in vitro. This precise and efficient ex vivo gene editing could provide a newly engineered cell line to improve the outcome of hMSC-based cell therapy. Figure 1 Funding Acknowledgement Type of funding source: Public hospital(s). Main funding source(s): The Foundation in Memory of Seymour Fefer, Sheba Medical Center

Non-alcoholic fatty liver disease (NAFLD) comprises a spectrum of progressive liver pathologies, ranging from simple steatosis to non-alcoholic steatohepatitis (NASH), fibrosis and cirrhosis. A liver biopsy is currently required to stratify high-risk patients, and predicting the degree of liver inflammation and fibrosis using non-invasive tests remains challenging. Here, we sought to develop a novel, cost-effective screening tool for NAFLD based on thermal imaging. We used a commercially available and non-invasive thermal camera and developed a new image processing algorithm to automatically predict disease status in a small animal model of fatty liver disease. To induce liver steatosis and inflammation, we fed C57/black female mice (8 weeks old) a methionine-choline deficient diet (MCD diet) for 6 weeks. We evaluated structural and functional liver changes by serial ultrasound studies, histopathological analysis, blood tests for liver enzymes and lipids, and measured liver inflammatory cell infiltration by flow cytometry. We developed an image processing algorithm that measures relative spatial thermal variation across the skin covering the liver. Thermal parameters including temperature variance, homogeneity levels and other textural features were fed as input to a t-SNE dimensionality reduction algorithm followed by k-means clustering. During weeks 3,4, and 5 of the experiment, our algorithm demonstrated a 100% detection rate and classified all mice correctly according to their disease status. Direct thermal imaging of the liver confirmed the presence of changes in surface thermography in diseased livers. We conclude that non-invasive thermal imaging combined with advanced image processing and machine learning-based analysis successfully correlates surface thermography with liver steatosis and inflammation in mice. Future development of this screening tool may improve our ability to study, diagnose and treat liver disease.

BACKGROUND: In vivo imaging of oxidative stress can facilitate the understanding and treatment of cardiovascular diseases. We evaluated nitroxide-enhanced MRI with 3-carbamoyl-proxyl (3CP) for the detection of myocardial oxidative stress. METHODS: Three mouse models of cardiac oxidative stress were imaged, namely angiotensin II (Ang II) infusion, myocardial infarction (MI), and high-fat high-sucrose (HFHS) diet-induced obesity (DIO). For the Ang II model, mice underwent MRI at baseline and after 7 days of Ang II (n = 8) or saline infusion (n = 8). For the MI model, mice underwent MRI at baseline (n = 10) and at 1 (n = 8), 4 (n = 9), and 21 (n = 8) days after MI. For the HFHS-DIO model, mice underwent MRI at baseline (n = 20) and 18 weeks (n = 13) after diet initiation. The 3CP reduction rate, K(red), computed using a tracer kinetic model, was used as a metric of oxidative stress. Dihydroethidium (DHE) staining of tissue sections was performed on Day 1 after MI. RESULTS: For the Ang II model, K(red) was higher after 7 days of Ang II versus other groups (p < 0.05). For the MI model, K(red), in the infarct region was significantly elevated on Days 1 and 4 after MI (p < 0.05), whereas K(red) in the noninfarcted region did not change after MI. DHE confirmed elevated oxidative stress in the infarct zone on Day 1 after MI. After 18 weeks of HFHS diet, K(red) was higher in mice after diet versus baseline (p < 0.05). CONCLUSIONS: Nitroxide-enhanced MRI noninvasively quantifies tissue oxidative stress as one component of a multiparametric preclinical MRI examination. These methods may facilitate investigations of oxidative stress in cardiovascular disease and related therapies.

Background and aim Exosome, the smallest vesicles that originate from intracellular compartments, contains and delivers specific bioactive proteins, lipids, mRNA, and miRNA to facilitate intercellular signaling. However, the role and mechanism of exosomes in atrial remodeling and fibrillation (AF) has not been determined. While epicardial fat (eFat) and inflammation have been linked to atrial remodeling and fibrillation (AF), the role of eFat exosomes in the pathogenesis of AF remains unknown. Thus, we aimed to determine whether eFat exosomes play a role in the initiation and progression of AF. Methods and results We collected eFat specimens from patients with and without chronic or paroxysmal AF undergoing heart surgery. Isolated fat specimens were cut into small pieces and incubated as organ cultures. We isolated exosomes from the medium of the explant by differential ultra-centrifugation and characterized the vesicle size distributions, morphology, specific markers, and molecular cargo. Immunoblotting of CD63, CD81 and TSG101 confirmed the presence of exosomes (Fig.1 B). Significantly, eFat from patients with AF secreted higher amounts of exosomes compared with controls (p Conclusion We show, for the first time, that exosomes from eFat of patients with AF have a unique pro-inflammatory and pro-fibrotic profile. We suggest a novel mechanism that links eFat to AF and eFat exosomes as novel biomarkers and potential therapeutic targets for AF.

Background Arteriogenesis is key for tissue repair but whether myocardial stromal cells contribute to this phenomenon remains unknown. Purpose Investigate if cardiac pericytes are a druggable target for therapeutic arteriogenesis. Methods and results The localization of pericyte-like cells (PCs) was assessed in the human and murine heart by immunohistochemistry of typical antigenic markers. CD34+ PCs co-expressing NG2 and PDGFRβ but not endothelial cell (EC) or vascular smooth muscle cell (VSMC) antigens were identified in peri-arterial position in normal hearts. Interestingly, we also found rare PCs co-expressing αSMA in the peri-infarct myocardium, suggesting these cells could represent a transitory phenotype between PCs and VSMCs. Next, we isolated human cardiac PCs by immunosorting for CD31 and CD34 and established the purity of the isolated CD34+CD31- fraction by flow cytometry. Following culture expansion, cardiac PCs maintain the typical antigenic profile except for CD34. Moreover, we confirmed the PCs' ability to promote angiogenesis in-vitro. The withdrawal of EGF and bFGF from the culture media for 10 days induced the differentiation of PCs into mature VSMCs, as documented by a massive upregulation of contractile genes MYH11, CNN1 and ACTA2 (200-, 35- and 15-folds increase versus naïve PCs, p Conclusions Cardiac PCs have a VSMC potential which is under the inhibitory control of the Mek1/2-Erk1/2-Elk1 signalling. Mek1/2 inhibitors showed promises for the treatment of melanoma and solid tumours. A novel application of this class of compounds to improve arteriogenesis in myocardial ischemia is fascinating. The caveat about their potential cardiotoxicity could be less relevant with short duration treatments of myocardial ischemia. Acknowledgement/Funding British Heart Foundation Centre for Regenerative Medicine Award (II) - “Centre for Vascular Regeneration” (RM/17/3/33381)

Purpose Thermal infrared imaging is a non-invasive tool with the potential to screen physiological processes and diseases. The use of this technique to image internal organs, such as the heart, has not yet been investigated. We aimed to determine the ability of a novel algorithm for thermal image-processing to detect structural and functional changes in a mouse model of cardiac remodeling. Methods We randomized 12 male mice (weight 20–25 gr) to treatment with either angiotensin-II (2 mg/kg/day, n=6) or saline (n=6) pump-infusion for 28 days. We measured blood pressure weekly, together with serial trans-thoracic echocardiography studies and histopathological evaluation of the hearts. We captured thermal images with the commercially available FLIR One camera, and processed images by our novel algorithm. Results Angiotensin infusion increased blood pressure together with cardiac hypertrophy and fibrosis. Thermal imaging identified an increase in the fraction of the skin heated by the heart in angiotensin-treated mice, at day 28 of the experiment. Thermal image findings were correlated to left ventricular mass and volume by echocardiography (r=0.6, p=0.07 and r=0.8, p Visual Abstract Conclusion Our preliminary findings suggest, for the first time, that a thermal camera with a new image-processing algorithm, identifies cardiac structural changes in mice. Our findings propose a new, simple, and non-invasive tool to diagnose and monitor adverse cardiac remodeling.

Introduction Macrophages control myocardial regeneration in the neonatal heart and repair in the adult heart. Osteopontin (OPN) is a macrophage-derived protein which is upregulated following tissue injury. While OPN regulates cell adhesion, spreading and migration, its role in myocardial regeneration is unknown. Purpose We aimed to test the hypothesis that OPN stimulates cardiomyocyte proliferation. Methods and results Mouse neonatal cardiomyocytes were treated with a serum-free medium for 24 hours. Immunofluorescent staining showed that these cells expressed the OPN receptor CD44 (Fig. 1A). To assess cell expansion, we used MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colorimetric assay. OPN preserved cardiomyocyte count in a low glucose medium for 24 hours, compared with no OPN treatment (Fig. 1B). Staining for phosphohistone (pH)3, a marker of nucleus division, showed a higher expression of pH3 in cardiomyocytes after OPN treatment (Fig. 1C). Immunoblotting revealed that OPN induced the phosphorylation of cell-cycle activity proteins, known as yes-associated protein (YAP)1, and extracellular signal-regulated kinase (ERK)2, while it decreased the phosphorylation of the YAP1 inhibitor, the large tumor-suppressor kinase (LATS)1/2 (Figs. 1D and 1E). Gene expression analysis revealed that OPN upregulated the transcriptional enhancer factor TEF-1 (TEAD1), which interacts with YAP1 (Fig. 1F), connective tissue growth factor (CTGF) and cyclin-dependent kinase 1 (CDK1), which are YAP downstream targets (Figs. 1G and 1H). Furthermore, OPN significantly upregulated cyclin B1, a regulator of the mitotic (M) phase (Fig. 1I). Moreover, OPN may promote positive feedback by upregulating the expression of CD44, SPP-1 (Figs. 1J and 1K). Finally, administration of OPN to adult mouse improved cardiac remodeling and function after myocardial infarction. Conclusions OPN stimulates cell-cycle activity in neonatal cardiomyocytes through YAP1 activity, independently from the HIPPO-YAP signaling pathway. Because macrophages are a major source of OPN after injury, our findings could explain the essential role of macrophages in neonatal heart regeneration and adult heart repair. Acknowledgement/Funding Israel Science Foundation

Background and Aim: Epicardial fat (eFat) has been linked to atrial remodeling and fibrillation (AF). We aimed to determine whether extracellular vesicles (EVs) derived from eFat play a role in the pathogenesis of AF. Methods and Results: We collected small specimens of eFat from patients (pts) with and without (w/o) AF undergoing heart surgery. eFat specimens were incubated as organ cultures and EVs were isolated from the culture medium by ultra-centrifugation. We used immunoblotting, electron microscopy, and nanoparticle tracking analysis to characterize the EVs ( Fig. A-C ). Significantly, eFat specimens from AF pts secreted greater amounts of EVs compared with patients w/o AF ( Fig. D ). Moreover, eFat EVs from AF pts secreted a higher concentration of inflammatory and fibrotic cytokines but less anti-inflammatory cytokines, compared with patients w/o AF ( Fig. E-H ). Notably, EV cytokines reflected the inflammatory and fibrotic status of eFat in AF pts better than free cytokines ( Fig. I-L ). Next, we tested several miRNA that could influence cardiac fibrosis. For example, miR-133 inhibits TGF-β, decreases collagen content and inhibits atrial remodeling. Expression of EV miR-133 was lower in eFat of AF pts than in patients w/o AF ( Fig. M ). Eventually, “wound healing” scratch assay show that fibroblast migration was greater after incubation with eFat EVs from AF patients, compared with pts w/o AF. ( Fig. N ). Conclusions: eFat from AF patients secretes a higher number of EVs with an inflammatory and fibrotic profile. Our findings suggest that eFat EVs contribute to inflammation and fibrosis, both of which contribute to the pathogenesis of atrial remodeling and fibrillation.

Many of the studies associating digoxin use with increased mortality were conducted before beta-blockers became a standard therapy for heart failure (HF) patients. Our goal was to determine the effect of beta-blockers on the prognosis of patients hospitalized for decompensated HF who receive digoxin therapy at discharge.We analyzed 2402 patients admitted with a primary diagnosis of decompensated HF during the prospective National Heart Failure Survey in Israel. Multivariate modeling was used to determine the effect of beta-blockers and digoxin on 1- and 10-year survival.Patients discharged on digoxin and beta-blockers (DIG+/BB+) had a lower 1-year mortality rate than those discharged on digoxin alone (DIG+/BB-), (31% vs. 44%; p0.001). Digoxin administration was associated with an increase in adjusted 1-year (Hazard ratio [HR] 1.28; 95% confidence interval (CI) 1.08-1.50) and 10-year mortality risk (HR 1.27; CI 1.16-1.42), whereas beta-blocker administration was associated with a decrease in adjusted 1-year (HR 0.76; CI 0.68-0.87) and 10-year mortality risk (HR 0.83; CI 0.77-0.89; all p0.001). In comparison to a DIG-/BB+ group serving as a reference, multivariate adjusted HR for DIG+/BB+ and DIG+/BB- groups were 1.36 (CI 1.03-1.91; p0.001) and 2.01 (CI 1.59-2.85; p0.001) at 1-year, and 1.04 (CI 0.84-1.28; p0.1) and 1.37 (CI 1.17-1.76; p0.001) at 10years.In patients hospitalized with decompensated HF, digoxin administration at discharge is associated with increased 1- and 10-year mortality risk. However, the simultaneous use of beta-blockers and digoxin is associated with lower 1- and 10-year mortality risk when compared to use of digoxin alone.

Despite improvements in modern cardiovascular therapy, the morbidity and mortality of ischaemic heart disease (IHD) and heart failure (HF) remain significant in Europe and worldwide. Patients with IHD may benefit from therapies that would accelerate natural processes of postnatal collateral vessel formation and/or muscle regeneration. Here, we discuss the use of cells in the context of heart repair, and the most relevant results and current limitations from clinical trials using cell-based therapies to treat IHD and HF. We identify and discuss promising potential new therapeutic strategies that include ex vivo cell-mediated gene therapy, the use of biomaterials and cell-free therapies aimed at increasing the success rates of therapy for IHD and HF. The overall aim of this Position Paper of the ESC Working Group Cellular Biology of the Heart is to provide recommendations on how to improve the therapeutic application of cell-based therapies for cardiac regeneration and repair.

Endothelial activation with up-regulation of E-selectin adhesion molecules mediates leukocyte rolling along the vascular wall and controls inflammation in many diseases including atherosclerosis and heart failure. Therefore, we aimed to test the hypothesis that inhibition of E-selectin-mediated interactions by a new E-selectin-targeted copolymer could inhibit the progression of atherosclerosis. To target E-selectin on activated endothelium, we developed a new N-(2-hydroxypropyl)methacrylamide (HPMA)-based E-selectin binding copolymer with or without dexamethasone (Dex) (designated P-(Esbp)-Dex and P-Esbp, respectively). To determine the effect of P-(Esbp)-Dex and P-Esbp on atherosclerosis, we allocated ApoE (−/−) mice on a high fat diet, to weekly intra-peritoneal injections of either 1) P-Esbp; 2) P-(Esbp)-Dex; 3) free Dex (1 mg/kg) or 4) saline, for four weeks. Aortic atherosclerosis and left ventricular (LV) remodeling and function were assessed by serial ultrasound studies and histology. Monocytes and macrophages were characterized by flow cytometry. After four weeks of treatment, P-Esbp effectively targeted aortic atherosclerotic lesions. Both P-Esbp and P-(Esbp)-Dex reduced wall thickening of the ascending aortas. However, only the drug-free copolymer (P-Esbp) significantly decreased the areas of necrotic core in the plaques and switched spleen macrophages toward an anti-inflammatory (M2) phenotype. Furthermore, P-Esbp attenuated adverse LV remodeling and dysfunction in ApoE (−/−) mice. In summary, P-Esbp copolymer targets activated endothelial cells, regresses and stabilizes atherosclerotic plaques, and prevents adverse LV remodeling and dysfunction in ApoE (−/−) mice. Our results suggest a new, drug-free macromolecular therapy to treat vascular inflammation.

The effectiveness of empagliflozin (EMPA), a sodium glucose cotransporter type 2 inhibitor, on the kidney, pancreas, and heart was investigated in the Cohen Rosenthal diabetic hypertensive rat model (CRDH rat). Six-week-old CRDH male rats were fed a sugar diet (SD) and treated with the compound EMPA (group Drug/SD) or respective comparator with vehicle (group Veh/SD). A control group was fed a regular diet without treatment (group Veh/P). Preventive treatment with EMPA was measured during 4 months of follow-up. The treatment effect was evaluated according to results observed after 4 months in group Drug/SD when compared to those in group Veh/SD. Significant effect resulted in the following parameters: enhancement of urinary glucose excretion in association with diuresis; amelioration of postprandial hyperglycemia and fasting blood glucose levels; and decrease in calculated Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) as well as lower systolic and diastolic blood pressures. At the end of treatment, EMPA preserved nephrin integrity in the kidney, reduced proteinuria, and prevented diabetes-induced damage to glomerular diaphragm structure. In the pancreas, EMPA demonstrated an impressive decrease in fatty infiltration and atrophy. Blood pressure was significantly reduced in the EMPA-treated group (15 ± 5.1 mm Hg, P < .05) in contrast to the vehicle and control groups. Finally, compared to controls, EMPA significantly reduced left ventricle (LV) mass and LV systolic dilatation, according to 2-dimensional echocardiography. The importance of the study lies in demonstrating the efficacy of an antidiabetic drug with beneficial effects on blood pressure, weight, kidney, and pancreas and a positive effect on the heart.

Idiopathic dilated cardiomyopathy (DCM) is a complex disorder with a genetic and an environmental component involving multiple genes, many of which are yet to be discovered. We integrate genetic, epigenetic, transcriptomic, phenotypic, and evolutionary features into a method – Hridaya, to infer putative functional genes underlying DCM in a genome-wide fashion, using 213 human heart genomes and transcriptomes. Many genes identified by Hridaya are experimentally shown to cause cardiac complications. We validate the top predicted genes, via five different genome-wide analyses: First, the predicted genes are associated with cardiovascular functions. Second, their knockdowns in mice induce cardiac abnormalities. Third, their inhibition by drugs cause cardiac side effects in human. Fourth, they tend to have differential exon usage between DCM and normal samples. Fifth, analyzing 213 individual genotypes, we show that regulatory polymorphisms of the predicted genes are associated with elevated risk of cardiomyopathy. The stratification of DCM patients based on cardiac expression of the functional genes reveals two subgroups differing in key cardiac phenotypes. Integrating predicted functional genes with cardiomyocyte drug treatment experiments reveals novel potential drug targets. We provide a list of investigational drugs that target the newly identified functional genes that may lead to cardiac side effects.

Korrespondenz zu 10.1093/cvr/cvx049

Background Patients with type 2 diabetes present with an accelerated atherosclerotic process. Animal evidence indicates that dipeptidyl peptidase-4 inhibitors (gliptins) have anti-inflammatory and anti-atherosclerotic effects, yet clinical data are scarcely available. Design and methods A prospective, randomized, open-label study was performed in 60 patients with coronary artery disease (CAD) and type 2 diabetes, who participated in a cardiac rehabilitation program. After a washout period of 3 weeks, patients were randomized in a 2:1 ratio to receive combined vildagliptin/metformin therapy (intervention group: n = 40) vs. metformin alone (control group: n = 20) for a total of 12 weeks. Blinded assessment of interleukin-1ß (IL-1ß, the primary endpoint), hemoglobin A1c (HbA1c), and high sensitivity C reactive protein (hsCRP), were performed at baseline and after 12 weeks. Results Mean age of study patients was 67 ± 9 years, 75% were males, and baseline HbA1c and inflammatory markers levels were similar between the two groups. At 12 weeks of follow up, levels of IL-1ß, hsCRP, and HbA1c were significantly lower in the intervention group as compared with the control group. There was a continuous elevation of IL-1ß among the control group, which was not observed in the intervention group (49 vs. 4%, respectively; p Conclusion The addition of vildagliptin to metformin treatment in patients with type 2 diabetes and CAD led to a significant suppression of the IL-1ß elevation during follow up. A significant relative reduction of hsCRP and HbA1c in the intervention group was also observed. Trial registration NCT01604213

Background The immune system plays a pivotal role in myocardial homeostasis and response to injury. Interleukins‐4 and ‐13 are anti‐inflammatory type‐2 cytokines, signaling via the common interleukin‐13 receptor α1 chain and the type‐2 interleukin‐4 receptor. The role of interleukin‐13 receptor α1 in the heart is unknown. Methods and Results We analyzed myocardial samples from human donors (n=136) and patients with end‐stage heart failure (n=177). We found that the interleukin‐13 receptor α1 is present in the myocardium and, together with the complementary type‐2 interleukin‐4 receptor chain Il4ra , is significantly downregulated in the hearts of patients with heart failure. Next, we showed that Il13ra1 ‐deficient mice develop severe myocardial dysfunction and dyssynchrony compared to wild‐type mice (left ventricular ejection fraction 29.7±9.9 versus 45.0±8.0; P =0.004, left ventricular end‐diastolic diameter 4.2±0.2 versus 3.92±0.3; P =0.03). A bioinformatic analysis of mouse hearts indicated that interleukin‐13 receptor α1 regulates critical pathways in the heart other than the immune system, such as extracellular matrix (normalized enrichment score=1.90; false discovery rate q=0.005) and glucose metabolism (normalized enrichment score=−2.36; false discovery rate q=0). Deficiency of Il13ra1 was associated with reduced collagen deposition under normal and pressure‐overload conditions. Conclusions The results of our studies in humans and mice indicate, for the first time, a role of interleukin‐13 receptor α1 in myocardial homeostasis and heart failure and suggests a new therapeutic target to treat heart disease.

International audience; Ischaemic heart disease and the heart failure that often results, remain the leading causes of death and disability in Europe and worldwide. As such, in order to prevent heart failure and improve clinical outcomes in patients presenting with an acute ST-segment elevation myocardial infarction and patients undergoing coronary artery bypass graft surgery, novel therapies are required to protect the heart against the detrimental effects of acute ischaemia/reperfusion injury (IRI). During the last three decades, a wide variety of ischaemic conditioning strategies and pharmacological treatments have been tested in the clinic-however, their translation from experimental to clinical studies for improving patient outcomes has been both challenging and disappointing. Therefore, in this Position Paper of the European Society of Cardiology Working Group on Cellular Biology of the Heart, we critically analyse the current state of ischaemic conditioning in both the experimental and clinical settings, provide recommendations for improving its translation into the clinical setting, and highlight novel therapeutic targets and new treatment strategies for reducing acute myocardial IRI.

Despite advances in myocardial reperfusion therapies, acute myocardial ischaemia/reperfusion injury and consequent ischaemic heart failure represent the number one cause of morbidity and mortality in industrialized societies. Although different therapeutic interventions have been shown beneficial in preclinical settings, an effective cardioprotective or regenerative therapy has yet to be successfully introduced in the clinical arena. Given the complex pathophysiology of the ischaemic heart, large scale, unbiased, global approaches capable of identifying multiple branches of the signalling networks activated in the ischaemic/reperfused heart might be more successful in the search for novel diagnostic or therapeutic targets. High-throughput techniques allow high-resolution, genome-wide investigation of genetic variants, epigenetic modifications, and associated gene expression profiles. Platforms such as proteomics and metabolomics (not described here in detail) also offer simultaneous readouts of hundreds of proteins and metabolites. Isolated omics analyses usually provide Big Data requiring large data storage, advanced computational resources and complex bioinformatics tools. The possibility of integrating different omics approaches gives new hope to better understand the molecular circuitry activated by myocardial ischaemia, putting it in the context of the human ‘diseasome’. Since modifications of cardiac gene expression have been consistently linked to pathophysiology of the ischaemic heart, the integration of epigenomic and transcriptomic data seems a promising approach to identify crucial disease networks. Thus, the scope of this Position Paper will be to highlight potentials and limitations of these approaches, and to provide recommendations to optimize the search for novel diagnostic or therapeutic targets for acute ischaemia/reperfusion injury and ischaemic heart failure in the post-genomic era.

Thermal infrared imaging has been suggested as a non-invasive alternative to monitor physiological processes and disease. However, the use of this technique to image internal organs, such as the heart, has not yet been investigated. We sought to determine the ability of our novel thermal image-processing algorithm to detect structural and functional changes in a mouse model of hypertension and cardiac remodeling. Twelve mice were randomly assigned to receive either the pro-inflammatory, hypertensive hormone angiotensin-II (2 mg/kg/day, n = 6) or saline (n = 6) infusion for 28 days. We performed weekly blood pressure measurements, together with serial trans-thoracic echocardiography studies and histopathological evaluation of the hearts. Thermal images were captured with a commercially available thermal camera, and images were processed by our novel algorithm which analyzes relative spatial temperature variation across the animal’s thorax. We assessed cardiac inflammation by measuring inflammatory cell infiltration through flow cytometry. Angiotensin infusion increased blood pressure together with cardiac hypertrophy and fibrosis. Thermal imaging at day 28 of the experiment detected an increase in the fraction of the skin heated by the heart in angiotensin-treated mice. Thermal image findings were significantly correlated to left ventricular volume and mass parameters seen on echocardiography (r = 0.8, p

Objectives This study sought to investigate the hypothesis that the favorable effects of mesenchymal stromal cells (MSCs) on infarct repair are mediated by macrophages. Background The favorable effects of MSC therapy in myocardial infarction (MI) are complex and not fully understood. Methods We induced MI in mice and allocated them to bone marrow MSCs, mononuclear cells, or saline injection into the infarct, with and without early (4 h before MI) and late (3 days after MI) macrophage depletion. We then analyzed macrophage phenotype in the infarcted heart by flow cytometry and macrophage secretome in vitro. Left ventricular remodeling and global and regional function were assessed by echocardiography and speckle-tracking based strain imaging. Results The MSC therapy significantly increased the percentage of reparative M2 macrophages (F4/80+CD206+) in the infarcted myocardium, compared with mononuclear- and saline-treated hearts, 3 and 4 days after MI. Macrophage cytokine secretion, relevant to infarct healing and repair, was significantly increased after MSC therapy, or incubation with MSCs or MSC supernatant. Significantly, with and without MSC therapy, transient macrophage depletion increased mortality 30 days after MI. Furthermore, early macrophage depletion produced the greatest negative effect on infarct size and left ventricular remodeling and function, as well as a significant incidence of left ventricular thrombus formation. These deleterious effects were attenuated with macrophage restoration and MSC therapy. Conclusions Some of the protective effects of MSCs on infarct repair are mediated by macrophages, which are essential for early healing and repair. Thus, targeting macrophages could be a novel strategy to improve infarct healing and repair.

Aims The aim of this study was to assess the use of a 3 T clinical cardiac magnetic resonance (CMR) scanner to detect injury to the heart in experimental autoimmune myocarditis (EAM). Methods and results The use of 3 T CMR for the detection of cardiac injury was assessed in EAM ( n = 55) and control ( n = 10) male Lewis rats. Animals were evaluated with serial CMR imaging studies, using a 3 T scanner, and with 2D echocardiography before, and at 2 and 5 weeks after EAM induction. By CMR, regional wall motion abnormalities were noted in seven out of eight rats with myocarditis 5 weeks after induction. Subsequently, the rats developed significant left ventricular (LV) dilatation, wall thickening, and pericardial effusion. Average LV systolic and diastolic volumes increased from 131 ± 10 to 257 ± 20 µL ( P = 0.0008), and from 309 ± 14 to 412 ± 24 µL ( P < 0.0001), and ejection fraction markedly deteriorated (from 58 ± 2 to 37 ± 5%; P = 0.0003). Areas of fibrosis were located by late gadolinium enhancement (LGE) CMR at the subepicardium, mainly within the anterior, lateral, and inferior walls. The extent and location of LGE were highly correlated ( r = 0.94; P < 0.0001) with areas of myocardial fibrosis by histopathology, with 85% sensitivity and 86% specificity. Conclusion A clinical 3 T CMR scanner enables accurate detection, quantification, and monitoring of experimental myocarditis in rats, and could be used for translational research to study the pathophysiology of the disease and evaluate novel therapies.

Background: Mesenchymal stromal cells (MSCs) improve tissue repair but their mechanism of action is not fully understood. We aimed to test the hypothesis that MSCs may act via macrophages, and that specifically, human cardiac adipose tissue-derived mesenchymal stromal cells (AT-MSCs) can polarize human macrophages into a reparative, anti-inflammatory (M2) phenotype. Methods and Results: We isolated and grew AT-MSCs from human cardiac adipose tissue obtained during cardiac surgery. Macrophages were grown from CD14+ monocytes from healthy donor blood and then cocultured with AT-MSCs, with and without transwell membrane, for 1 to 14 days. In response to AT-MSCs, macrophages acquired a star-shaped morphology, typical of alternatively activated phenotype (M2), and increased the expression of M2 markers CD206+, CD163+, and CD16+ by 1.5- and 9-fold. Significantly, AT-MSCs modified macrophage cytokine secretion and increased the secretion of anti-inflammatory and angiogenic cytokines: interleukin (IL)-10 (9-fold) and vascular endothelial growth factors (3-fold). Moreover, AT-MSCs decreased macrophage secretion of inflammatory cytokines such as IL-1α (2-fold), tumor necrosis factor α (1.5-fold), IL-17 (3-fold), and interferon gamma (2-fold). Remarkably, the interaction between AT-MSCs and macrophages was bidirectional and macrophages enhanced AT-MSC secretion of typical M2 inducers IL-4 and IL-13. Notably, AT-MSCs decreased macrophage phagocytic capacity. Finally, IL-6 mediates the M2 polarization effect of AT-MSCs on macrophages, by increasing M2-associated cytokines, IL-10 and IL-13. Conclusions: Human cardiac AT-MSCs can polarize human macrophages into anti-inflammatory phenotype. Our findings suggest a new mechanism of action of AT-MSCs that could be relevant to the pathogenesis and treatment of myocardial infarction, atherosclerosis, and various cardiovascular diseases.

The murine neonatal heart can regenerate after injury through cardiomyocyte (CM) proliferation, although this capacity markedly diminishes after the first week of life. Neuregulin-1 (NRG1) administration has been proposed as a strategy to promote cardiac regeneration. Here, using loss- and gain-of-function genetic tools, we explore the role of the NRG1 co-receptor ERBB2 in cardiac regeneration. NRG1-induced CM proliferation diminished one week after birth owing to a reduction in ERBB2 expression. CM-specific Erbb2 knockout revealed that ERBB2 is required for CM proliferation at embryonic/neonatal stages. Induction of a constitutively active ERBB2 (caERBB2) in neonatal, juvenile and adult CMs resulted in cardiomegaly, characterized by extensive CM hypertrophy, dedifferentiation and proliferation, differentially mediated by ERK, AKT and GSK3β/β-catenin signalling pathways. Transient induction of caERBB2 following myocardial infarction triggered CM dedifferentiation and proliferation followed by redifferentiation and regeneration. Thus, ERBB2 is both necessary for CM proliferation and sufficient to reactivate postnatal CM proliferative and regenerative potentials.

Uncontrolled activation of pro-inflammatory macrophages after myocardial infarction (MI) accelerates adverse left ventricular (LV) remodeling and dysfunction. Hemin, an iron-containing porphyrin, activates heme oxygenase-1 (HO-1), an enzyme with anti-inflammatory and cytoprotective properties. We sought to determine the effects of hemin formulated in a macrophage-targeted lipid-based carrier (denoted HA-LP) on LV remodeling and function after MI. Hemin encapsulation efficiency was ~ 100% at therapeutic dose levels. In vitro, hemin/HA-LP abolished TNF-α secretion from macrophages, whereas the same doses of free hemin and drug free HA-LP had no effect. Hemin/HA-LP polarized peritoneal and splenic macrophages toward M2 anti-inflammatory phenotype. We next induced MI in mice and allocated them to IV treatment with hemin/HA-LP (10 mg/kg), drug free HA-LP, free hemin (10 mg/kg) or saline, one day after MI. Active in vivo targeting to infarct macrophages was confirmed with HA-LP doped with PE-rhodamine. LV remodeling and function were assessed by echocardiography before, 7, and 30 days after treatment. Significantly, hemin/HA-LP effectively and specifically targets infarct macrophages, switches infarct macrophages toward M2 anti-inflammatory phenotype, improves angiogenesis, reduces scar expansion and improves infarct-related regional function. In conclusion, macrophage-targeted lipid-based drug carriers with hemin switch macrophages into an anti-inflammatory phenotype, and improve infarct healing and repair. Our approach presents a novel strategy to modulate inflammation and improve infarct repair.

Background: Little is known about the potentially unfavorable effects of mesenchymal stromal cell (MSC) activation on the heart. MSCs can respond to tissue injury by anti- or proinflammatory activation. We aimed to study the potential negative interaction between left ventricular dysfunction (LVD) and MSC activation. Methods: We isolated MSCs from cardiac and subcutaneous fat tissues of mice with LVD 28 days after myocardial infarction or sham operation. To evaluate the effect of LVD on MSCs, we characterized cardiac MSCs and subcutaneous MSCs in vitro. Subsequently, we injected MSCs or saline into the infarcted myocardium of mice and evaluated LV remodeling and function 28 days after myocardial infarction. To test the hypothesis that toll-like receptor 4 ( TLR4 ) mediates proinflammatory polarization of MSCs, we characterized cardiac MSCs from TLR4 -/- and wild-type (WT) mice after inflammatory stimulation in vitro. Next, we transplanted cardiac MSCs from TLR4 -/- and WT male mice into the infarcted myocardium of female WT mice and evaluated infarct size, MSC retention, inflammation, remodeling, and function after 7 days. Results: LVD switched cardiac MSCs toward an inflammatory phenotype, with increased secretion of inflammatory cytokines as well as chemokines. The effect of LVD on subcutaneous MSCs was less remarkable. Although transplantation of cardiac MSCs and subcutaneous MSCs from LVD and sham hearts did not improve LV remodeling and function, cardiac MSCs from LVD exacerbated anterior wall thinning 28 days after myocardial infarction. The inflammatory polarization of cardiac MSCs by LVD was mediated by TLR4 , as we found less secretion of inflammatory cytokines and higher secretion of anti-inflammatory cytokines from activated cardiac MSCs of TLR4 -deficient mice, compared with WT cardiac MSCs. Significantly, TLR4 deficiency preserved the expression of CD47 (don’t eat me signal) on cardiac MSCs after both TLR4 stimulation in vitro and transplantation into the infarcted heart. Compared with WT cardiac MSCs and saline, TLR4 -/- cardiac MSCs survived in the cardiac tissue and maintained their reparative properties, reduced infarct size, increased scar thickness, and attenuated LV dilatation 7 days after myocardial infarction. Conclusions: The environment of the failing and infarcted myocardium drives resident and transplanted MSCs toward a proinflammatory phenotype and restricts their survival and reparative effects in a mechanism mediated by TLR4 .

Nowadays, combination therapy became a standard in oncology. In this study, we compare the activity of two polymeric carriers bearing a combination of the anticancer drugs paclitaxel (PTX) and doxorubicin (DOX), which differ mainly in their architecture and supramolecular assembly. Drugs were covalently bound to a linear polymer, polyglutamic acid (PGA) or to a dendritic scaffold, polyglycerol (PG) decorated with poly(ethylene glycol) (PEG), forming PGA-PTX-DOX and PG-PTX-bz-DOX-PEG, respectively. We explored the relationship between the polymeric architectures and their performance with the aim to augment the pharmacological benefits of releasing both drugs simultaneously at the tumor site at a synergistic ratio. We recently designed and characterized a PGA-PTX-DOX conjugate. Here, we describe the synthesis and characterization of PG dendritic scaffold bearing the combination of PTX and DOX. The performance of both conjugates was evaluated in a murine model of mammary adenocarcinoma in immunocompetent mice, to investigate whether the activity of the treatments is affected by the immune system. Drug conjugation to a nano-sized polymer enabled preferred tumor accumulation by extravasation-dependent targeting, making use of the enhanced permeability and retention (EPR) effect. Both PGA-PTX-DOX and PG-PTX-bz-DOX-PEG nano-sized conjugates exhibited superior anti-tumor efficacy and safety compared to the combination of the free drugs, at equivalent concentrations. However, while PGA-PTX-DOX was more efficient than a mixture of each drug conjugated to a separate PGA chain, as was previously shown, PG-PTX-bz-DOX-PEG had similar activity to the mixture of the PG-PTX-bz-PEG and PG-DOX-PEG conjugates. Our results show that both conjugates are potential candidates as precision combination nanomedicines for the treatment of breast cancer.

Background Irreversible electroporation (IRE) is a non-thermal cell ablation approach that induces selective damage to cell membranes only. The purpose of the current study was to evaluate and optimize its use for in-vivo myocardial decellularization. Methods Forty-two Sprague-Dawley rats were used to compare myocardial damage of seven different IRE protocols with anterior myocardial infarction damage. An in-vivo open thoracotomy model was used, with two-needle electrodes in the anterior ventricular wall. IRE protocols included different combinations of pulse lengths (70 vs. 100 μseconds), frequency (1, 2, 4 Hz), and number (10 vs. 20 pulses), as well as voltage intensity (50, 250 and 500 Volts). All animals underwent baseline echocardiographic evaluation. Degree of myocardial ablation was determined using repeated echocardiography measurements (days 7 and 28) as well as histologic and morphometric analysis at 28 days. Results All animals survived 28 days of follow-up. Compared with 50V and 250V, electroporation with 500V was associated with significantly increased myocardial scar and reduction in ejection fraction (67.4%±4% at baseline vs. 34.6%±20% at 28 days; p

To test the hypothesis that magnetic resonance (MR) imaging R1 (R1 = 1/T1) mapping after selectively labeling monocytes with a T1-shortening contrast agent in vivo would enable the quantitative measurement of their spatiotemporal kinetics in the setting of infarct healing.All procedures were performed in mice and were approved by the institutional committee on animal research. One hundred microliters of dual-labeled liposomes (DLLs) containing gadolinium (Gd)-diethylenetriaminepentaacetic acid (DTPA)-bis(stearylamide) and DiI dye were used to label monocytes 2 days before myocardial infarction (MI). MI was induced by occlusion of the left anterior descending coronary artery for 1 hour, followed by reperfusion. MR imaging R1 mapping of mouse hearts was performed at baseline on day -3, on day 0 before MI, and on days 1, 4, and 7 after MI. Mice without labeling were used as controls. ΔR1 was calculated as the difference in R1 between mice with labeling and those without labeling. CD68 immunohistochemistry and DiI fluorescence microscopy were used to confirm that labeled monocytes and/or macrophages infiltrated the postinfarct myocardium. Statistical analysis was performed by using two-way analysis of variance and the unpaired two-sample t test.Infarct zone ΔR1 was slightly but nonsignificantly increased on day 1, maximum on day 4 (P.05 vs all other days), and started to decrease by day 7 (P.05 vs days -3, 0, and 1) after MI, closely reflecting the time course of monocyte and/or macrophage infiltration of the infarcted myocardium shown by prior histologic studies. Histologic results confirmed the presence and location of DLL-labeled monocytes and/or macrophages in the infarct zone on day 4 after MI.R1 mapping after labeling monocytes with T1-shortening DLLs enables the measurement of post-MI monocyte and/or macrophage spatiotemporal kinetics.

Herein we investigated a new strategy for the modulation of cardiac macrophages to a reparative state, at a predetermined time after myocardial infarction (MI), in aim to promote resolution of inflammation and elicit infarct repair. The strategy employed intravenous injections of phosphatidylserine (PS)-presenting liposomes, mimicking the anti-inflammatory effects of apoptotic cells. Following PS-liposome uptake by macrophages in vitro and in vivo, the cells secreted high levels of anti-inflammatory cytokines [transforming growth factor β (TGFβ) and interleukin 10 (IL-10)] and upregulated the expression of the mannose receptor—CD206, concomitant with downregulation of proinflammatory markers, such as tumor necrosis factor α (TNFα) and the surface marker CD86. In a rat model of acute MI, targeting of PS-presenting liposomes to infarct macrophages after injection via the femoral vein was demonstrated by magnetic resonance imaging (MRI). The treatment promoted angiogenesis, the preservation of small scars, and prevented ventricular dilatation and remodeling. This strategy represents a unique and accessible approach for myocardial infarct repair.

Proper spatio-temporal delivery of multiple therapeutic proteins represents a major challenge in therapy strategies aimed at inducing myocardial regeneration after myocardial infarction (MI). We hypothesized that the dual delivery of insulin-like growth factor-1 (IGF-1) and hepatocyte growth factor (HGF) by injectable affinity-binding alginate biomaterial would maximize their therapeutic effects, leading to a more favorable course of tissue restoration after acute MI. A sequential release of IGF-1 followed by HGF was attained from affinity-binding alginate biomaterial, which also protected the proteins from proteolysis (shown by mass spectroscopy). The released factors retained bioactivity, as judged by their capability to activate their respective signaling pathways and to prevent cardiomyocyte apoptosis in vitro. In a rat model of acute MI, an intramyocardial injection of the dual IGF-1/HGF affinity-bound alginate biomaterial preserved scar thickness, attenuated infarct expansion and reduced scar fibrosis after 4 weeks, concomitantly with increased angiogenesis and mature blood vessel formation at the infarct. Furthermore, this treatment prevented cell apoptosis, induced cardiomyocyte cell cycle re-entry and increased the incidence of GATA-4-positive cell clusters. The dual delivery of IGF-1 and HGF from affinity-binding alginate biomaterial represents a useful strategy to treat MI. It showed a marked therapeutic efficacy at various tissue levels, as well as potential to induce endogenous regeneration of cardiac muscle.